CN104880525B - LC-MS for criminal investigation purpose detects the method for zolpidem poisoning label phenyl-4-carboxylic acid zolpidem in urine - Google Patents
LC-MS for criminal investigation purpose detects the method for zolpidem poisoning label phenyl-4-carboxylic acid zolpidem in urine Download PDFInfo
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Abstract
The open method of zolpidem poisoning label phenyl 4 carboxylic acid zolpidem in the LC-MS detection urine of criminal investigation purpose of the present invention, comprises the steps: that (1) obtains liquid to be detected after being processed by urine sample；(2) the phenyl 4 carboxylic acid zolpidem in the liquid to be detected that Liquid Chromatography-Tandem Mass Spectrometry combined instrument detecting step (1) obtains is utilized.The method of inspection of the present invention has advantage highly sensitive, that detection limit is low；And it is simple to operate, the response rate is high, and selectivity is strong, can be used for the contamination test sensitivity of zolpidem in urine, thus improves the detection of zolpidem poisoning Related Cases in the inspection of poisoning and criminal case.
Description
Technical field
The present invention relates to the poisonous substance detection method in criminal investigation field, detect urine particularly to the LC-MS for criminal investigation purpose
The method of middle zolpidem poisoning label phenyl-4-carboxylic acid zolpidem.
Background technology
Zolpidem is one of representative medicine of third generation hypnotic-non-benzodiazepine sedative hypnotic drugs, and trade name Stilnox, for miaow
Azoles pyridines hypnotic.Along with zolpidem application clinically increases, forensic science field also occurs in that relevant case
Part.Owing to it absorbs fast, metabolism is fast, and Check-Out Time is short, and substance is difficult to detection.Therefore, research zolpidem and metabolite thereof, bright
Really bioconversion rule, significant for case inspection and forensic science etc..
Zolpidem chemistry entitled N, N, 6-trimethyl-2-(4-tolyl) imidazo [1,2-a]-pyridine-3-acetamide, point
Minor C19H21N3O, molecular weight 307, structural formula is as shown in formula I.The therapeutic dose that medicine is recommended is 5～10mg, and it is administered orally
Absorbing rapidly, have the highest plasma protein binding rate (92.5%), can pass through blood brain barrier, cerebrospinal fluidconcentration is blood concentration
30～50%, its metabolite parmacodynamics-less activity, the former medicine only less than 1% is discharged from urine.Zolpidem in vivo main
Metabolite is 6-carboxylic acid zolpidem (Zolpidem6-carboxylic acid, ZCA) and phenyl-4-carboxylic acid zolpidem
(Zolpidem phenyl-4-carboxylic acid, ZPCA), structural formula is respectively as shown in formula II and formula III.
After zolpidem entrance is internal, receptor binding site there is is high selectivity, produce obvious hypnosis after taking medicine calm
Effect, side effect and untoward reaction are the least many.Although this kind of safety of medicine scope is relatively big, but during large dose oral administration also causes
Poison.Poisoning manifestations is in various degree drowsiness, dizzy, weak, ataxia, feels sick, and vomiting shows as individually excitement, multi-lingual.
Severe patient can cause stupor, blood pressure drops, shock, and breath cycle suppresses, dead etc..Single new sleeping drug toxicity is less,
Oral a large amount of zolpidem acute poisonings death, often can detect other sedative hypnotics or antidepressant drug or ethanol simultaneously,
The most not can determine that the definite toxic dose of this type of medicine and lethal dose data.
In toxicological analysis is identified, occur that some utilize zolpidem to implement to commit suiside, anaesthetize robbery, anesthesia homicide, fiber crops in recent years
The liquor-saturated criminal case raped.Due to the metabolic characteristic that the rapid-action elimination of this medicine is fast, propose higher requirement to trace detection.
The elimination half-life of zolpidem is the shortest, and only 0.7～3.5h, detection of seldom having an opportunity after therefore taking 24～48h
Zolpidem.In anesthesia robbery, urine is one of the most frequently used sample sample, owing to poisonous substance has longer in urine
Check-Out Time window, therefore urine has more peculiar advantage than blood or serum etc..But, also not for urine in prior art
The detection method of phenyl-4-carboxylic acid zolpidem in liquid, particular without detection method highly sensitive, that detection limit is low.
Summary of the invention
In view of this, the invention reside in a kind of highly sensitive, LC-MS for criminal investigation purpose that detection limit is low is provided
The method of zolpidem poisoning label phenyl-4-carboxylic acid zolpidem in detection urine.
The present invention is achieved through the following technical solutions: in the LC-MS detection urine of criminal investigation purpose in zolpidem
The method of poison label phenyl-4-carboxylic acid zolpidem, comprises the steps:
(1) liquid to be detected is obtained after being processed by urine sample；
(2) phenyl-4-carboxylic in the liquid to be detected that Liquid Chromatography-tandem Mass instrument detecting step (1) obtains is utilized
Acid zolpidem.
Said method, in step (2), the chromatographic column of liquid chromatograph is: a) chromatographic column: Agilent Eclipse Plus
C18RRHD post, 2.1mm × 100mm, 1.7 μm；Or b) chromatographic column: ACQUITY UPLC HELIC post, 2.1mm × 100mm,
1.7μm。
Said method, in step (2), the flowing of liquid chromatograph is any one combination following mutually:
Flow combined one: by A phase and B phase composition, A phase is water, and B phase is acetonitrile；
Flow combined two: by A phase and B phase composition, A phase be the volume fraction of acetonitrile be 0.05%, the volume integral of formic acid
Number is 0.1% and the mixed solution 2A that volume fraction is 99.85% of water；B phase be the volume fraction of water be 0.05%, formic acid
Volume fraction is 0.1% and the mixed solution 2B that volume fraction is 99.85% of acetonitrile；
Flow combined three: by A phase and B phase composition, A phase be the volume fraction of water be 95% and formic acid acetonitrile solution A
Volume fraction is the mixed solution 3A of 5%, and wherein in formic acid acetonitrile solution A, the volume fraction of formic acid is 1%, and mixes molten
Liquid 3A is also added into the ammonium acetate solution that ammonium acetate concentration is 20mmol/L；B phase be the volume fraction of water be 5% and formic acid second
The volume fraction of nitrile solution B is the mixed solution 3B of 95%, and wherein in formic acid acetonitrile solution B, the volume fraction of formic acid is
1%.
Said method, in step (2), the eluent gradient elution requirement of liquid chromatograph is following three kinds of eluent gradients
Any one in elution requirement:
Eluent gradient elution requirement one, illustrates with the ratio of Mobile phase B: initial volume 10% keeps 0.4min
After, 2.2min increases to 50% holding 0.3min, and 2.6min drops to 10% and keeps 2min；
Eluent gradient elution requirement two, illustrates with the ratio of Mobile phase B: initial volume 10% keeps 0.4min
After, 1.5min increases to 90% holding 1min, and 2.6min drops to 10% and keeps 2min；
Eluent gradient elution requirement three, illustrates with the ratio of Mobile phase B: initial volume 10% keeps 0.5min
After, 1.5min slowly increases to 70% holding 1min, and 2.6min drops to 10% and keeps 2min.
Said method, in step (2), the sample size of liquid chromatograph is 1 μ L, flow velocity is 0.4mL/min.
Said method, in step (2), Mass Spectrometry Conditions is: electric spray ion source ESI, ionization mode: ESI+；Scanning
Pattern: multiple-reaction monitoring MRM；Ion source voltage: 5500V；Source temperature: 550 DEG C；Gas curtain gas: 30psi, atomization gas: 60psi, auxiliary
Help gas: 55psi, remove a bunch voltage: 150.
Said method, in step (1), urine sample processing method is as follows: take urine sample, adds acetonitrile, and mixing is shaken
Swing, centrifugal, take supernatant and be liquid to be detected through organic membrane filter, gained filtrate.
Said method, takes urine sample, adds acetonitrile, mixing vibration, is centrifuged, takes supernatant and cross organic filter membrane, the most again
CrossPLD+ removes removing protein and phospholipid 96 orifice plate, and negative pressure evacuation crosses organic filter membrane, and gained filtrate is to be detected
Liquid.
Said method, in step (1), urine sample processing method is as follows: take urine sample, adds acetate esters chemical combination
Thing, 1, in 1-dimethyl acetone and diallyl ether any two or three, mixing vibration, stratification, separate aqueous phase and have
Machine phase；Any two kinds or three in acetate compounds, 1,1-dimethyl acetone and diallyl ether are added again in aqueous phase
Kind, again mix vibration, stratification and separate aqueous phase and organic facies, merging organic facies；Organic facies after merging is placed in dense
Evaporate into dry on contracting instrument, residue methanol constant volume, with organic membrane filtration and in being transferred to sample injection bottle for detection.
Said method, described acetate compounds is ethyl acetate or methyl acetate
The invention has the beneficial effects as follows: the acetonitrile precipitation albumen-dephosphorization of the phenyl-4-carboxylic acid zolpidem that the present invention sets up
Fat-liquid chromatography/mass spectrometry method of inspection, simple to operate, detection limit is low, and the response rate is high, and selectivity is strong, can be used for azoles pyrrole in urine
Smooth contamination test sensitivity, thus improve the inspection of zolpidem poisoning Related Cases in the inspection of poisoning and criminal case
Survey.
Accompanying drawing explanation
Fig. 1 is Agilent Eclipse Plus C18 column chromatography figure.
Fig. 2 is the chromatogram of UPLC HELIC post.
Fig. 3 is the chromatogram of the first flowing phase.
Fig. 4 is the chromatogram of the second flowing phase.
Fig. 5 is the chromatogram of the third flowing phase.
Fig. 6 is blank liquid matter MRM figure.
Fig. 7 be 3 mix target liquid matter MRM figure.
Detailed description of the invention
For understanding the scheme in the explanation present invention, preferred embodiment is given below and is described with reference to the accompanying drawings.
Embodiment 1
The present embodiment is zolpidem poisoning label phenyl-4-carboxylic acid azoles pyrrole in the LC-MS detection urine of criminal investigation purpose
Smooth method, comprises the steps:
One, experiment reagent, instrument and experimental facilities
1 experiment reagent
1.1 standard substance and reagent
Phenyl-4-carboxylic acid zolpidem standard substance, content 99wt%, it is purchased from Toronto chemical company of Canada；
Formic acid, acetonitrile, ammonium acetate, methanol etc. are chromatographically pure reagent, purchased from traditional Chinese medicines group；
Ultra-pure water is prepared through Millipore simplicity pure water system.
1.2 standard solution and the preparation of related solvents
The preparation of standard reserving solution: accurately weigh 10mg phenyl-4-carboxylic acid zolpidem standard substance and be dissolved in methanol, and constant volume
To scale, it is configured to the standard substance storing solution of 1mg/mL, 4 DEG C of refrigerators preserve, standby.
The preparation of standard working solution: take the standard reserving solution prepared, be diluted to 0.1 successively, 0.01,0.001mg/mL system
The working solution of row concentration, preserves in 4 DEG C of refrigerators, standby.
0.1% aqueous formic acid: 0.5mL formic acid ultra-pure water is diluted to 500mL.
2 materials
PLD+ removes removing protein and phospholipid 96 orifice plate: phosphide plate is removed in 96 holesPLD+50mg 96
Orifice plate (Biotage company of Sweden).
Blank sample: urine, picks up from the healthy volunteer not taking medicine.
3 instruments and experimental facilities
API 5500 Q-TRAP MS/MS (Applied Biosystems company, the U.S.), is equipped with electric spray ion source
(ESI), triple quadrupole bar mass analyzer.
Shimadzu 30A-LC (Shimadzu company, Japan), is equipped with Shimadzu 30A automatic sampler
(Shimadzu company, Japan).
BP210s electronic balance (Sartorius company of Germany)；Millipore SimLicity purification of water system (method
State)；Scientific Industries vortex oscillator (USA)；SORVALL High speed refrigerated centrifuge (German)；EYELA is high
Speed agitator (Japanese).
Two, obtain liquid to be detected after being processed by urine sample: take urine 0.2mL, add 0.8mL acetonitrile, mixing vibration
10min, 8000rpm are centrifuged 10min, take supernatant 200 μ L and filter through organic membrane filter (the organic filter membrane of micropore: 0.22 μm), gained
Liquid is liquid to be detected, liquid to be detected is placed on concentrating instrument evaporate into dry, residue methanol constant volume, with organic membrane filtration also
Detection is supplied in being transferred to sample injection bottle.
Three, the zolpidem in the liquid to be detected that Liquid Chromatography-tandem Mass instrument detecting step (two) obtains is utilized.
The chromatographic column of liquid chromatograph is: a) chromatographic column: Agilent Eclipse Plus C18RRHD post, 2.1mm ×
100mm, 1.7 μm.
The flowing of liquid chromatograph is mutually for flowing combined: by A phase and B phase composition, A phase is water, and B phase is acetonitrile.
The eluent gradient elution requirement of liquid chromatograph is eluent gradient elution requirement one, enters with the ratio of Mobile phase B
Row explanation (as shown in table 1): after initial volume 10% keeps 0.4min, 2.2min increases to 50% holding 0.3min, under 2.6min
It is down to 10% and keeps 2min.
Table 1 condition of gradient elution one
| Time (min) | Flow velocity (mL/min) | Mobile phase A (V%) | Mobile phase B (V%) |
| 0.1 | 0.4 | 90 | 10 |
| 0.4 | 0.4 | 90 | 10 |
| 2.2 | 0.4 | 50 | 50 |
| 2.5 | 0.4 | 50 | 50 |
| 2.6 | 0.4 | 90 | 10 |
| 4.6 | 0.4 | 90 | 10 |
The sample size of liquid chromatograph is 1 μ L, flow velocity is 0.4mL/min.
Mass Spectrometry Conditions is: electric spray ion source ESI, ionization mode: ESI+；Scan pattern: multiple-reaction monitoring MRM；From
Component voltage: 5500V；Source temperature: 550 DEG C；Gas curtain gas: 30psi, atomization gas: 60psi, assist gas: 55psi, remove a bunch voltage:
150.Detection ion pair, collision energy is shown in Table 2.
Table 2 detects ion pair, impact energy scale
Embodiment 2
Embodiment 2 is with the difference of embodiment 1, and chromatographic column is: ACQUITY UPLC HELIC post, 2.1mm ×
100mm,1.7μm。
Embodiment 3
Embodiment 3 is with the difference of embodiment 1, and the flowing of liquid chromatograph is mutually for flowing combined two: by A phase and B phase
Composition, A phase be the volume fraction of acetonitrile be 0.05%, the volume fraction of formic acid is 0.1% and the volume fraction of water is 99.85%
The volume fraction that mixed solution 2A, B phase is water be 0.05%, the volume fraction that volume fraction is 0.1% and acetonitrile of formic acid
It is the mixed solution 2B of 99.85%.
Embodiment 4
Embodiment 4 is with the difference of embodiment 1, and the flowing of liquid chromatograph is mutually for flowing combined three: by A phase and B phase
Composition, A phase be the volume fraction of water be 95% and the mixed solution 3A, Qi Zhong that volume fraction is 5% of formic acid acetonitrile solution A
In formic acid acetonitrile solution A, the volume fraction of formic acid is 1%, and it is dense to be also added into ammonium acetate in every 100 milliliters of mixed solution 3A
Degree is the ammonium acetate solution 0.5-2 milliliter of 20mmol/L；B phase be the volume fraction of water be 5% and the volume of formic acid acetonitrile solution B
Mark is the mixed solution 3B of 95%, and wherein in formic acid acetonitrile solution B, the volume fraction of formic acid is 1%.
Embodiment 5
Embodiment 5 is with the difference of embodiment 1, and the eluent gradient eluting of liquid chromatograph is eluent gradient eluting bar
Part two, illustrates (as shown in table 3) with the ratio of Mobile phase B: after initial volume 10% keeps 0.4min, 1.5min increases to
90% keeps 1min, 2.6min drop to 10% and keep 2min.
Table 3 condition of gradient elution two
| Time (min) | Flow velocity (mL/min) | Mobile phase A (V%) | Mobile phase B (V%) |
| 0.1 | 0.4 | 90 | 10 |
| 0.4 | 0.4 | 90 | 10 |
| 1.5 | 0.4 | 10 | 90 |
| 2.5 | 0.4 | 10 | 90 |
| 2.6 | 0.4 | 90 | 10 |
| 4.6 | 0.4 | 90 | 10 |
Embodiment 6
Embodiment 6 is with the difference of embodiment 1, and the eluent gradient eluting of liquid chromatograph is eluent gradient eluting bar
Part three, illustrates (as shown in table 4) with the ratio of Mobile phase B: after initial volume 10% keeps 0.5min, 1.5min slowly increases
1min, 2.6min is kept to drop to 10% and keep 2min to 70%.
Table 4 condition of gradient elution three
| Time (min) | Flow velocity (mL/min) | Mobile phase A (V%) | Mobile phase B (V%) |
| 0.1 | 0.4 | 90 | 10 |
| 0.5 | 0.4 | 90 | 10 |
| 1.5 | 0.4 | 30 | 70 |
| 2.5 | 0.4 | 30 | 70 |
| 2.6 | 0.4 | 90 | 10 |
| 4.6 | 0.4 | 90 | 10 |
Embodiment 7
Embodiment 7 is with the difference of embodiment 1, and in step (1), urine sample processing method is as follows: take urine
0.2mL, adds 0.8mL acetonitrile, and mixing vibration 10min, 8000rpm are centrifuged 10min, take supernatant organic through the micropore of 0.22 μm
Membrane filtration, then afterPLD+ removes removing protein and phospholipid 96 orifice plate, and negative pressure evacuation crosses 0.22 μm again
Organic filter membrane, gained filtrate is liquid to be detected, for Instrumental Analysis.
Embodiment 8
Embodiment 7 is with the difference of embodiment 1, and in step (1), urine sample processing method is as follows: take 5mL urine
Sample, adds methyl acetate and 1, mixed solution 5mL (methyl acetate and 1, the volume of 1-dimethyl acetone of 1-dimethyl acetone
Ratio is 3:2), mixing vibration, stratification, separate aqueous phase and organic facies；Methyl acetate and 1,1-diformazan is added again in aqueous phase
The mixed solution 5mL (methyl acetate and 1, the volume ratio of 1-dimethyl acetone is 3:2) of benzylacetone, again mixes vibration, stands
It is layered and separates aqueous phase and organic facies, merge organic facies；Will merge after organic facies be placed on concentrating instrument evaporate into dry, residue use
Methanol constant volume, with organic membrane filtration and in being transferred to sample injection bottle for detection.
Embodiment 9
Embodiment 7 is with the difference of embodiment 1, and in step (1), urine sample processing method is as follows: take 5mL urine
Sample, adds ethyl acetate and the mixed solution 5mL (ethyl acetate and diallyl ether volume ratio are 3:2) of diallyl ether,
Mixing vibration, stratification, separate aqueous phase and organic facies；The mixing adding ethyl acetate and diallyl ether again in aqueous phase is molten
Liquid 5mL (ethyl acetate and diallyl ether volume ratio are 3:2), again mixes vibration, stratification and separates aqueous phase and organic
Phase, merges organic facies；Will merge after organic facies be placed on concentrating instrument evaporate into dry, residue methanol constant volume, use organic filter membrane
Detection is supplied in being filtered and transferred to sample injection bottle.
Embodiment 10
Embodiment 7 is with the difference of embodiment 1, and in step (1), urine sample processing method is as follows: take 5mL urine
Sample, add ethyl acetate, 1, the mixed solution 5mL of 1-dimethyl acetone and diallyl ether (ethyl acetate, 1,1-dimethyl
The volume ratio of acetone and diallyl ether is 1.5:1.5:2), mixing vibration, stratification, separate aqueous phase and organic facies；Again to
Aqueous phase adds ethyl acetate, 1,1-dimethyl acetone and mixed solution 5mL (ethyl acetate, the 1,1-diformazan of diallyl ether
The volume ratio of benzylacetone and diallyl ether is 1.5:1.5:2), again mix vibration, stratification and separate aqueous phase and organic
Phase, merges organic facies；Will merge after organic facies be placed on concentrating instrument evaporate into dry, residue methanol constant volume, use organic filter membrane
Detection is supplied in being filtered and transferred to sample injection bottle.
Experimental result and the relative analysis of embodiment 1-embodiment 10 are as follows:
1, the optimized choice of mass spectrometry parameters
Accurately weigh phenyl-4-carboxylic acid zolpidem standard substance, become the list mark standard solution of 20ng/mL with acetontrile, adopt
By pin pump form direct injected, it is first determined the mass number of object parent ion.On this basis selection intensity maximum front 3～
4 daughter ions, compare optimization to collision energy, by gradually changing collision energy, make parent ion intensity account for maximum son
The 1/3～1/4 of ionic strength.2 daughter ions that final selected intensity is the highest, form two to mother/daughter ion pair as qualitative point
The index of analysis, using the strongest ion pair as quantitative analysis index.Two pairs of mother/daughter ions that successive optimization is selected on this basis
Remove a bunch voltage, collision energy, inject voltage, collision cell injection voltage etc., it is thus achieved that optimum complete mass spectrometric data.
The optimization of 2 liquid-phase conditions
The optimization of 2.1 chromatographic columns
In the optimization of liquid phase post, embodiment 1 and embodiment 2 compare Agilent Eclipse Plus C18RRHD
(2.1mm × 100mm, 1.7 μm) and ACQUITY UPLC HELIC (2.1mm × 100mm, 1.7 μm) post.The former is for 3 mesh
Standard substance and the interpolation separation of sample, the chromatographic peak peak shape of mark thing are all preferable (see Fig. 1), but the latter's HELIC post presents separation
The best, chromatographic peak trails, and has bigger substrate to affect (see Fig. 2) on it.
The optimization of 2.2 flowing phases
In terms of flowing phase, embodiment 1, embodiment 3 and embodiment 4 compare that flowing is combined one, flows combined two
The impact of three these three flowing relative separation detections combined with flowing.Result shows, the effect using these three flowing phase is equal
Preferably (see Fig. 3～5), chromatographic peak peak shape is good, but flows combined two and the sound of the combined three pairs of 6-carboxylic acid zolpidems that flow
Should be less, zolpidem and the 6-carboxylic acid zolpidem retention time in combined three of flowing is nearer；Flow zolpidem, benzene in combined two
The retention time difference maximum of base-4-carboxylic acid zolpidem and 6-carboxylic acid zolpidem three.Therefore, comprehensive factors above selects simple
Efficient flowing combined is as flowing phase.
The optimization of 2.3 gradients
Embodiment 1, embodiment 5 and embodiment 6 compare different eluent gradient condition, finally determine that eluent gradient is washed
De-condition one is optimum condition, it can be ensured that object separates good, retention time stable (as shown in Figure 3).
2.3 specificity
Under the instrument condition that embodiment 1 is selected, zolpidem and 6-carboxylic acid zolpidem, phenyl-4-carboxylic acid zolpidem have
Good chromatographic peak.Under this chromatographic condition, analysis margin urine sample is noiseless, shows that the method has preferable specificity, chromatographic peak
Peak shape and separate good (Fig. 6-7).
2.4 sample treatment
2.4.1 acetonitrile precipitation albumen and mistakePLD+ removes removing protein and phospholipid 96 orifice plate
PLD+ goes the effect of removing protein and phospholipid 96 orifice plate to be phosphide and deproteinization, embodiment 7
In after acetonitrile precipitation albumen, afterPLD+ removes removing protein and phospholipid 96 orifice plate.Result shows, mistakePLD+ goes the average recovery rate after removing protein and phospholipid 96 orifice plate to return than the average of simple acetonitrile precipitation albumen
Yield the best (being shown in Table 5), illustrate after acetonitrile precipitation albumen afterPLD+ goes removing protein and phospholipid 96 orifice plate to help
In reducing in biological material the substrate impacts on object such as phosphide, the phenyl-4-carboxylic acid zolpidem response rate can be improved.
Table 5 acetonitrile precipitation albumen and mistakePLD+ removes removing protein and the average recovery rate of phospholipid 96 orifice plate
2.4.2 organic solvent extraction
Acetonitrile precipitation albumen and mistakePLD+ removes removing protein and phospholipid 96 orifice plate, although can reduce biology
The substrate impact on object such as phospholipid in sample, but the phenyl-4-carboxylic acid zolpidem response rate is the most relatively low.Embodiment 8-is real
Executing the method that example 10 uses mixed organic solvents to extract, phenyl-4-carboxylic acid zolpidem average recovery rate is above 90%, Qi Zhongshi
Execute the average recovery rate in example 10 and reach as high as more than 97% (being shown in Table 6).
The average recovery rate of table 6 organic solvent extraction
2.5 standard curves and sensitivity
Standard reserving solution initial flow phase dilution is become 0.10ng/mL, 1.00ng/mL, 5.00ng/mL, 10.00ng/
Phenyl-4-carboxylic acid zolpidem series standard the liquid of mL, 50.00ng/mL, instrument sample introduction 1 μ L analyzes.With concentration of standard solution as horizontal stroke
Coordinate, chromatographic peak area is that vertical coordinate does standard linear regression, obtains regression equation.Phenyl-4-carboxylic acid zolpidem series standard
Liquid is: y=46443x-3615.4；Correlation coefficient is R2=0.9999.Normal linearity scope is 0.1ng/mL～50ng/mL, spirit
Sensitivity is 0.08ng/mL.
2.6 working curves and detection limit
Take blank urine samples 5 parts, each 0.2mL respectively, add phenyl-4-carboxylic acid zolpidem series standard liquid and be prepared as adding
Urine sample, makes solution final concentration of 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, uses embodiment 7 or real
Execute the method for inspection in example 10, carry out Instrumental Analysis.With concentration, corresponding peak area is carried out linear regression, obtain regression equation
For: y=40806x-18733；In 1ng/mL～the 50ng/mL range of linearity, correlation coefficient is R2=0.9983.By signal/noise ratio
(S/N) >=3, calculate detection in urine and be limited to 0.1ng/mL.
2.7 response rate and precision
Take blank urine samples 0.2mL, add phenyl-4-carboxylic acid zolpidem series standard liquid and be prepared as adding urine sample,
Make solution final concentration of 1ng/mL, 5ng/mL, 50ng/mL, each concentration operation repetitive 5 parts.Use the urine sample in embodiment 7
Product processing method, chromatographic condition and Mass Spectrometry Conditions carry out Instrumental Analysis.The peak area recorded and the face, standard substance peak of corresponding concentration
Long-pending ratio is as the response rate, and average recovery rate is 90.8%.
Take blank urine samples 0.2mL, add phenyl-4-carboxylic acid zolpidem series standard liquid and be prepared as adding urine sample,
Make solution final concentration of 1ng/mL, 5ng/mL, 50ng/mL, each concentration operation repetitive 5 parts.Use the urine in embodiment 10
Sample treatment, chromatographic condition and Mass Spectrometry Conditions carry out Instrumental Analysis.The peak area recorded and the standard substance peak of corresponding concentration
The ratio of area is as the response rate, and average recovery rate is 97.7%.
The sample preparing high, medium and low 3 kinds of concentration detects, and uses at the urine sample in embodiment 7 or embodiment 10
Reason method, chromatographic condition and Mass Spectrometry Conditions detect.Parallel 5 parts of each concentration, examines at interior 3 different times
Surveying, calculate the relative standard deviation of object peak area, obtain withinday precision, two object results are respectively less than 5%.According to same
The sample of quadrat method 3 kinds of concentration of preparation carries out extraction detection, surveys 1 batch every day, surveys 5d continuously, calculates object peak area and relatively marks
Quasi-deviation, obtains day to day precision, and two object results are respectively less than 10% (being shown in Table 7).
The precision table of table 7 phenyl-4-carboxylic acid zolpidem
By embodiment 1-embodiment 10 it can be seen that the acetonitrile precipitation of phenyl-4-carboxylic acid zolpidem set up of the present invention
Albumen-go phospholipid or the liquid chromatography/mass spectrometry method of inspection of organic solvent extraction, simple to operate, detection limit is low, and the response rate is high,
Selectivity is strong, can be used for the contamination test sensitivity of zolpidem in urine, thus improves in poisoning and the inspection of criminal case
The detection of middle zolpidem poisoning Related Cases.
In sum, the method for the present invention can be used in biological sample the qualitative analysis of phenyl-4-carboxylic acid zolpidem and fixed
Component analysis.Qualitative analysis: if occurring identical with phenyl-4-carboxylic acid zolpidem standard working solution retention time in biological specimen
Chromatographic peak, enter and also generate and the mass spectrum of the phenyl-4-corresponding molecular weight of carboxylic acid zolpidem after mass spectrum, then show target to be measured
Containing phenyl-4-carboxylic acid zolpidem in biological specimen.Quantitative analysis: make with the drafting identical chromatographic condition of standard curve
Go out the chromatogram of phenyl-4-carboxylic acid zolpidem in target organism sample to be measured, measure chromatographic peak area and peak height, then according to peak
Area and peak height directly find in injection chromatographic column phenyl-4-carboxylic acid zolpidem in target organism sample to be measured on standard curve
Concentration.
Above-described embodiment is only for clearly demonstrating the invention example, and not has the invention
The restriction of body embodiment.For those of ordinary skill in the field, can also make on the basis of the above description
The change of other multi-form or variation.Here without also cannot all of embodiment be given exhaustive.All the present invention's
Any obvious change extended out or change the guarantor still in the invention claim within spirit and principle
Protect among scope.
Claims (7)
1. use zolpidem poisoning label phenyl-4-carboxylic acid azoles in liquid chromatography-tandem mass spectrometry detection urine for criminal investigation purpose
The method that pyrrole is smooth, it is characterised in that comprise the steps: that (1) obtains liquid to be detected after being processed by urine sample；(2) liquid is utilized
Phenyl-4-carboxylic acid zolpidem in the liquid to be detected that phase chromatograph-tandem mass spectrum combined instrument detecting step (1) obtains；Liquid chromatograph
Chromatographic column be: Agilent Eclipse Plus C18 RRHD post, 2.1mm × 100mm, 1.7 m；The flowing of liquid chromatograph
By A phase and B phase composition, A phase is water, and B phase is acetonitrile；The eluent gradient elution requirement of liquid chromatograph: with the ratio of Mobile phase B
Example illustrates, and after initial volume 10% keeps 0.4min, 2.2min increases to 50% holding 0.3min, and 2.6min drops to 10% also
Keep 2min.
Method the most according to claim 1, it is characterised in that in step (2), the sample size of liquid chromatograph is 1 μ L, stream
Speed is 0.4mL/min.
3. according to the arbitrary described method of claim 1-2, it is characterised in that in step (2), Mass Spectrometry Conditions is: electron spray
Ion source ESI, ionization mode: ESI+；Scan pattern: multiple-reaction monitoring MRM；Ion source voltage: 5500V；Source temperature: 550
℃；Gas curtain gas: 30psi, atomization gas: 60psi, assist gas: 55psi, remove a bunch voltage: 150.
Method the most according to claim 3, it is characterised in that in step (1), urine sample processing method is as follows: take
Urine sample, adds acetonitrile, mixing vibration, is centrifuged, takes supernatant and be liquid to be detected through organic membrane filter, gained filtrate.
Method the most according to claim 3, it is characterised in that take urine sample, adds acetonitrile, mixing vibration, is centrifuged, takes
Supernatant crosses organic filter membrane, then removes removing protein and phospholipid 96 orifice plate after ISOLUTE PLD+, and negative pressure evacuation is the most organic
Filter membrane, gained filtrate is liquid to be detected.
Method the most according to claim 3, it is characterised in that in step (1), urine sample processing method is as follows: take
Urine sample, add acetate compounds, 1, in 1-dimethyl acetone and diallyl ether any two or three, mixing is shaken
Swing, stratification, separate aqueous phase and organic facies；Acetate compounds, 1,1-dimethyl acetone and two is added again in aqueous phase
In allyl ether any two or three, again mixes vibration, stratification and separates aqueous phase and organic facies, merging organic
Phase；Organic facies after merging is placed on concentrating instrument and evaporates into dry, and residue methanol constant volume, with organic membrane filtration and be transferred to
For detection in sample injection bottle.
Method the most according to claim 6, it is characterised in that described acetate compounds is ethyl acetate or acetic acid first
Ester.
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Citations (2)
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|---|---|---|---|---|
| US20070027180A1 (en) * | 2005-09-19 | 2007-02-01 | Padi Pratap R | Process for preparing zolpidem |
| CN101277959A (en) * | 2005-10-03 | 2008-10-01 | 马林克罗特公司 | Process for preparing zolpidem hemitartrate and tartrate polymorphs |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070027180A1 (en) * | 2005-09-19 | 2007-02-01 | Padi Pratap R | Process for preparing zolpidem |
| CN101277959A (en) * | 2005-10-03 | 2008-10-01 | 马林克罗特公司 | Process for preparing zolpidem hemitartrate and tartrate polymorphs |
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|---|
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| A Simple and Rapid Method for the Identification of Zolpidem Carboxylic Acid in Urine;John H. Lewis 等;《Journal of Analytical Toxicology》;20070531;第31卷;第195-199页 * |
| CYP3A4 and CYP2C19 genetic polymorphisms and zolpidem metabolism in the Chinese Han population: A pilot study;Min Shen 等;《Forensic Science International》;20120908;第227卷;第77-81页 * |
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